Expression Patterns of E-Cadherin and N-Cadherin Proteins in the Periodontal Pocket Epithelium of Chronic Periodontitis

Statement of the Problem: E-cadherin and N-cadherin are two types of cell adhesion molecules that are involved in organ development, wound healing, and pathological conditions through the process of epithelial-mesenchymal transition (EMT). However, their role has not yet been fully elucidated in the pathogenesis of periodontal diseases. Purpose: To determine the expression level of proteins associated with the EMT process (E-cadherin and N-cadherin) in chronic periodontitis. Materials and Method: In this cross-sectional study, 37 samples (19 cases with healthy gingival tissue and 18 cases with severe chronic periodontitis) that referred to the Periodontology Department of Zahedan Dental School, Zahedan, Iran, in 2018 were included. The samples were immunohistochemically stained with E-cadherin and N-cadherin monoclonal antibodies. Afterward, the percentage of stained cells and the staining intensity of the cells were evaluated. Finally, the obtained data were analyzed using by IBM© SPSS© Statistics version 21 using Mann-Whitney statistical test. Results: In this study, 89.5% of the healthy gingival tissue samples and 61.1% of samples with chronic periodontitis showed E-cadherin expression in more than 50% of cells. This difference between the two groups was not significant (p= 0.13); however, the E-cadherin staining intensity of the healthy gingival tissue was strong while that of the samples with chronic periodontitis was moderate (p= 0.002). The N-cadherin expression was negative in 68.4% of healthy gingival cases, while 50% of the cases with chronic periodontitis showed a high expression of N-cadherin. This difference was statistically significant (p= 0.04). Moreover, the N-cadherin staining intensity also had a significant difference between the two groups (p= 0.004). Conclusion: Based on the results of the present study, the increased expression of N-cadherin and reduction of staining intensity of E-cadherin was found in chronic periodontitis compared to healthy gingival tissues. Therefore, EMT process may be involved in the pathogenesis of severe chronic periodontitis.


Introduction
Periodontitis is the sixth most prevalent disease worldwide and is related to oral squamous cell carcinoma [1].
It is an inflammatory and polymicrobial disease which affects the supporting structure of tooth and it is determined by a lack of epithelial attachment and alveolar bone resorption, which can lead to tooth loss if not treated properly [1][2]. According to the World Health Organization (WHO), 35-50% of the world population suffers from periodontitis [3].
Junctional and sulcular epithelium are the first barriers versus the dental biofilms, which act as mechanical barriers, they provide the local immune response by discharging inflammatory cytokines and enzymes. Lack of epithelial integrity can be associated with bacterial invasion to the lower connective tissue and potentially increase the inflammatory response and subsequent injury [4].
Epithelial-mesenchymal transition (EMT) is a rapid and often reversible change from epithelial cell phenotype. This process is essential for proper development during embryogenesis and pathological conditions, such as degenerative fibrosis and cancer [5][6].
During this process, cell phenotypes change from epithelial to mesenchymal, a process that is essentially characterized by the loss of cellular junctions, an increase in mesenchymal markers and a decrease in epithelial markers [6]. According to previous studies, chronic and persistent inflammation caused by microbial infections can induce EMT in different organs [7][8].
It is also possible that EMT starts in the epithelial cells of the mouth during chronic inflammation and leads to the loss of epithelial attachment and epithelial barrier function, and thereby the increase of microbial infiltration in the gingiva and peri-radicular tissues.
However, the potential and the exact role of EMT in periodontal diseases, as a mechanism that participates in the epithelial barrier function and oral bacterial infiltration, are not exactly determined [9]. E-cadherin is a glycoprotein belonging to the cadherin family. It plays an important role in epithelial cohesion through the establishment of epithelial cellular junctions and its absence increases the epithelial cells motility and their ability of local invasion; it is known as an EMT marker in tumor progression. [10][11].
N-cadherin (CDH2) is a calcium-dependent adhesion glycoprotein from the cadherin family. This protein is located on the 18q11 chromosome and normally occurs in neuroectodermal and mesodermal tissues. It plays an essential role in the establishment of cellular adhesions in mesenchymal cells. Many studies have indicated the re-expression, increased expression, or decreased N-cadherin expression in human neoplasms and tumor cell categories, especially breast, prostate and thyroid cancer [11][12][13][14][15]. The epithelial cells do not express N-cadherin; however, they could obtain it through cadherin switching process, a process that occurs during EMT and increases cell mobility and invasive properties [10]. The term of cadherin switching commonly refers to the switching from E-cadherin expression to N-cadherin expression [16].
Only few researches have been performed on the role of EMT in the pathogenesis of periodontal diseases. Abdulkareem et al. [2] found that long-term exposure of rat oral keratinocytes cultures to periodontal pathogens created EMT-like features; this process might have a role in epithelial integrity loss during periodontitis. However, the role of this process in the pathogenesis of periodontal diseases is not exactly determined [2]. Therefore, the current study aimed to determine the role of EMT in severe chronic periodontitis through the E-cadherin and N-cadherin proteins expression.
Intensity of staining was scored as negative (none staining), mild (light brown staining of the cells), severe (dark brown staining of the cells), and moderate (between mild and severe staining of the cells) [17].

Statistical Analysis
Data analysis was carried out using IBM© SPSS© Statistics version 21 (IBM© Corp., Armonk, NY, USA).
The relationship between the groups was assessed by Mann-Whitney test. p Value less than 0.05 were considered statistically significant.

Results
The mean ages of the subjects with healthy gingiva and  (Figures 1-2). Tables 3 and 4 also show the staining intensity of the cells towards E-cadherin and Ncadherin in the studied groups.

Discussion
Maintaining the cohesion of the epithelium performs an  Oral epithelium responds to the periodontal pathoge-ns by secretion of cytokines and chemokines such as tumor necrosis factor (TNF-α), transforming growth factor-β1 (TGF-β1), and epidermal growth factor, which trigger the beginning of EMT [7]. The effect of EMT in the pathogenesis of periodontitis has not yet been extensively studied. Therefore, the current study investigated the expression of EMT markers (E-cadherin and Ncadherin) in healthy gingiva and chronic periodontitis.
According to the results of the present study, no significant decrease in E-cadherin expression was observed in chronic periodontitis compared to healthy gingival group, but the staining intensity was severe in the majo-   found a reduction in E-cadherin expression in response to stimulation by periodontal pathogens. This process has often been attributed to the gram-negative bacterium P.gingivalis [2]. The results of a study performed by Nagarakanti et al. [18], revealed proteolytic degradation  [20]. In addition, monolayer epithelium permeability in tissue culture increased associated with reduction of Ecadherin expression due to exposure to lipopolysaccharide (LPS) of P.gingivalis [21]. These data are inconsistent with our study that showed no significant decrease in the E-cadherin expression protein in chronic periodntitis tissues, compared to healthy gingival tissues. This is probably due to small sample size and applying sensitive IHC technique in our study. In addition, sample processing and primary antibodies may affect the results.
Microbial pathogens play an essential role in the induction of EMT signaling pathways and mesenchymal phenotype changes. For example, the Helicobacter Pylori organism activates EMT through the MAPK/ErK-NF-kB pathways and the suppression of GSK3β activity in gastric cancer. In a study performed by Lee et al. [22], it was found that P.gingivalis, as an opportunistic pathogen in prolonged infections in the oral epithelium, was strongly associated with the induction of early changes in EMT. These changes begin with an increase in GSK-3β phosphorylation which, in turn, increases the expression of transcription factors (i.e., Snail, Slug, and Zeb1), followed by a loss and decrease of E-cadherin expression with B-catenin accumulation in oral epithelial cells. These molecular events appear to increase the mesenchymal phenotype and migration in epithelial cells.
In the current study, a considerable increase in Ncadherin expression was found in chronic periodontitis tissues, compared to healthy gingival tissues. There was also a statistically significant difference between the two groups in terms of cell staining intensity towards Ncadherin, which was consistent with the findings of Abdulkareem et al. study [2]. In their study, cultured epithelial cells were exposed to periodontal pathogens (F.